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Human Journals

Research Article

November 2017 Vol.:8, Issue:1

© All rights are reserved by D.R. Tiku et al.

Palm Oil Mill Effluent Biodegradation Potentials among Local

Isolated Microorganisms

www.ijsrm.humanjournals.com

Keywords: Palm Oil, Biodegradation, Microorganisms

ABSTRACT

The research study was aimed at investigating palm oil mill

biodegradation potentials among locally isolated

microorganisms. Palm Oil Mill Effluent (POME) polluted

soil samples (0-15cm and 15-30cm depth) were collected into

sterile polythene bags from Uruk Etta Ikot in Etim Ekpo

L.G.A and Eyop industries at Akamkpa L.G.A, while POME

wastewater was collected from Mfamosing at Akamkpa

L.G.A, into glass containers with Teflon lined cap. The

samples were placed in an ice-cool chest and transported to

the laboratory for analysis. The study was completed within a

duration of six months. Standard microbiological techniques

were used to isolate, characterize and identify isolates from

the collected samples. The biodegradation potentials of the

isolates were adjudged based on their ability to reduce

Biological Oxygen Demand (BOD), Chemical Oxygen

Demand (COD), Oil and grease and Total Suspended Solids

(TSS) of the palm oil mill effluents. The results obtained from

the study showed that the BOD of the raw POME samples

were reduced from 45,000±3.44mg/l to 37000±2.57mg/l

(Bacillus spp), 36100±2.77mg/l (Micrococcus spp),

35,100mg/l (Pseudomonas spp), 35,200±3.08mg/l

(Aspergillus spp), 33,400±2.54mg/l (Fusarium spp) and

25,600±1.81mg/l (Mucor spp). However the COD, Oil and

grease, and TSS reduction potentials of the isolates followed

similar pattern, with Pseudomonas spp (29,000±1.87mg/l),

showing the highest COD reduction potential and Mucor spp

(1,800±0.27mg/l, and 11,121±1.41mg/L) showing the highest

oil and grease and TSS reduction potential respectively after a

duration of 5 days. However, from this study, it is a clear

indicator that Pseudomonas spp (with 53.33% BOD and

47.37% COD reduction efficiency), Aeromonas spp, (57.24%

oil and grease reduction potential). Fusarium spp and Mucor

spp (with 64% oil and grease reduction potential) and Mucor

spp (23.20% TSS reduction potential) could be useful in

enhancing the bioremediation and treatment of POME

polluted environments.

E.O. Eno, S.P. Antai, *D.R. Tiku

Department of Microbiology, University of Calabar,

Calabar

Submission: 19 October 2017

Accepted: 29 October 2017

Published: 30 November 2017


Citation: D.R. Tiku et al. Ijsrm.Human, 2017; Vol. 8 (1): 53-65.

54

INTRODUCTION

Palm oil industry has become one of the main agro-industries in Nigeria. Palm oil mills

release POME in colossal amounts with its attendant polluting impending (Mohammadreza et

al., 2014). POME has unfavorable environmental ramifications effects including land and

aquatic ecosystem contamination, loss of biodiversity and increase in COD and BOD in

environment (Wu et al., 2010). Today, the penetration of palm oil has been considered due to

the entry of its effluents into the waterways and ecosystems, making it a meticulous concern

towards the food chain interference and water consumption (Foo and Hameed et al., 2010).

This can cause considerable environmental problems such as land pollution and effectively

suffocating aquatic life if discharged without effective treatment (Cheng et al., 2010). In the

process of palm oil milling, POME is mainly generated from sterilization and clarification of

palm oil, in which a large amount of steam and hot water are used (Ohimain et al., 2010).

POME is a thick brownish liquid that is compiled with high concentrations of total solids, oil

and grease, chemical oxygen demand (COD) and biological oxygen demand (BOD) (Neoh et

al., 2013). The biological treatment of POME depends enormously on consortium of

microbial activities, which operate on the organic substrates present in the POME as

supplements and eventually degrade these organic matters into simple by product such as

methane, carbon dioxide hydrogen and water (Mohammed et al., 2014). A variety of

microorganisms have been investigated to be capable of biodegrading oil wastewater with

high profits. Although, the anaerobic and aerobic treatment are one of the most sort biological

methods for POME treatment. However the suspended and colloidal components are neither

effectively decomposed biologically nor by other conventional means because they float the

surface of wastewater and this the major problem to cause failure of treatment system

(Ahmad et al., 2011). However, with due consideration of the aforementioned, this study

intends to investigate POME biodegradation potentials among locally isolated

microorganisms.

MATERIALS AND METHODS

POME polluted soil samples:

POME polluted soil samples for this study was obtained from Uruk Etta Ikot in Etim Ekpo

L.G.A and Eyop industries at Akamkpa L.G.A. The site was chosen with the intent of using

soil samples that probably had in the past been exposed to POME.



55

Collection:

Surface soil (0 to 15cm depth) and subsurface soil (15cm-30cm depth) were collected using a

Dutch auger. Collection was made from 3 to 4 random point, then bulked to form a composite

sample and deposited in sterile labeled polyethylene bag and transported to the laboratory for

further analysis.

POME wastewater

POME wastewater was collected into glass containers with Teflon lined cap from Eyop

industries and Mfamosing, both located at Akamkpa L.G.A. The samples were placed in an

ice-cool chest and transported to laboratory for analysis.

Media:

The media used in the study were; nutrient agar (Oxoid Ltd.), Sabouraud Dextrose Agar

(Oxoid Ltd.) and Tributyrin agar (Oxoid Ltd.). The media were prepared in accordance to the

manufacture instructions.

METHODS

Isolation of isolates

The method used was that of (Antai et al., 2016). In this method, surface- spreading

technique was used. Ten fold serial dilution of the POME polluted soil samples and

wastewater samples were prepared. 0.1ml of 10-6

dilution was plated into nutrient agar and

sabouraud dextrose agar plates. The nutrient agar plates were supplemented with 50µgml-1

of

nystatin so as to inhibit fungal growth whole the sabouraud dextrose agar plates were

supplemented with100µml-1

of streptomycin, so as to inhibit bacterial growth. The plates

were prepared in duplicates and incubated at 37ºC for 24hours (nutrient agar plate) and 28ºC

for 72 hours (sabouraud dextrose agar plate).

Purification and maintenance of microbial isolates

The bacterial and fungal isolates obtained from the nutrient agar and sabouraud dextrose agar

plates were purified by repeated sub-culturing. The isolates were subjected to series of

transfer into newly prepared medium. The bacteria isolates were transferred into newly

prepared nutrient agar medium and incubated at 37ºC for 24 hours. The fungal isolates,



56

however, were transferred into newly prepared sabouraud dextrose agar and incubated at

28ºC for 3 days. Pure colonies of the bacteria and fungi were maintained on slopes of nutrient

agar and sabouraud dextrose agar slant and stored in a refrigerator at 8ºC.

Characterization and identification of the isolates

Standard inocula were prepared from the preserved stock culture by taking a loopful of the

bacterial and fungal isolates and aseptically inoculating onto sterile nutrient agar and

sabouraud dextrose agar respectively. The plates were incubated at 28ºC for 24 hours. The

characterization of the isolates was then performed by employing gram staining reaction,

biochemical characterization and microscopic examination (Holt et al., 1994; Cheebrough

2000; Hunter and Bennet, 1993).

Biodegradation test

250ml of raw palm oil mill effluent was introduced into conical flash and sterilized at 121ºC

for 20 minutes. The sterilized raw palm oil mill effluent was allowed to cool before

inoculation. 20mls of broth sample containing the inoculums was used to inoculate 250mls of

the palm oil mil effluent samples and was shaken and incubated at room temperature for 24

hours (bacteria) and 3-4 days (fungi). The samples were aseptically drawn for 5 days and

analyzed for BOD, COD, oil and grease and TSS, with the control flasks not inoculated. The

percentage reduction was measured by using the equation as described by (Jeremiah et al.,

2014).

Reduction (%) =

Where;

Craw POME is the concentration of COD, BOD, TSS, OIL and grease of raw POME and cf

is the concentration of these parameters after treatment.

RESULTS

Palm oil mill effluent biodegradation potential by isolates from collection sample.

Table 1 presents result of POME biodegradation potential of selected isolates from collected

samples. It showed that Bacillus spp (PCE1) was able to reduce the BOD of the raw POME



57

from 45,000 2.87mg/l to 37,000±2.87mg/l after a duration of 5 days reduced while BOD

value of 36,100±2.77mg/l, 22,100±1.57mg/l, 29,700±2.18mg/l, 35,600±1.81mg/l were

obtained from Micrococcus spp (PSC1), Pseudomonas spp (PSC3), Aeromonas spp (PSC6),

Geotricum spp (PSU18), Aspergillus spp (PSC5), Fusarium spp (PSC18) and Mucor spp

(PUE6) respectively (fig 1). The COD of the POME was reduced from 55,100±3.89 to

40,501±3.21mg/l (Bacillus spp), 46,123±3.14mg/l (Micrococcus spp), 29,000±1.87mg/l

(Pseudomonas spp), 40,100±3.15mg/l (Aeromonas spp), 45,200±2.56mg/l (Geotricum spp)

44,200 ±2.98 (Aspergillus spp), 43,100±2.89mg/l (Fusarium spp) and 38,400±2.05mg/l

(Mucor spp) (fig 2). However, the oil and grease and TSS of the palm oil mill effluent

substrate followed similar pattern (fig 3 and 4).

Table 1: Palm oil mill effluent biodegradation potential of isolated isolates from

collected samples

Isolate

code

Numbe

r of

days

BOD(mg/L) COD(mg/L) Oil and

Grease(mg/L)

TSS(mg/L) Probable

organism

PCE1 1

2

3

4

5

45,000

40,300

38,700

41,500

37,400

55,100

37,470

48,000

46,700

40,501

5,000

4,560

4,100

4,000

3,800

14,480

14,300

13,800

13,200

12,900

Bacillus spp

PSC1 1

2

3

4

5

45,000

38,210

36,500

39,301

36,100

55,100

50,300

51,105

48,000

46,123

5,000

4,900

3,700

3,520

3,300

14,480

14,100

13,900

13,540

12,000

Micrococcus

spp

PSC3 1

2

3

4

5

45,000

35,200

28,800

25,240

21,000

55,100

35,190

33,000

30,100

29,000

5,000

3,400

3,180

2,190

1,900

14,480

14,101

14,068

13,780

13,660

Pseudomonas

spp

PSC6 1

2

3

4

5

45,000

28,800

24,200

30,500

29,700

55,100

42,200

39,420

37,260

40,100

5,000

3,200

3,100

3,080

2,138

14,480

14,098

14,053

13,590

12,300

Aeromonas

spp



58

PSU18 1

2

3

4

5

45,200

38,700

36,100

33,200

35,100

55,100

53,200

52,600

48,100

45,200

5,000

4,000

3,800

3,420

3,200

14,480

13,121

13,011

12,281

12,211

Geotricum

spp

PCE5 1

2

3

4

5

45,300

44,100

41,300

38,400

35,200

55,100

52,300

49,300

49,100

44,200

5,000

3,600

3,400

3,200

3,100

14,480

14,112

14,001

13,411

13,010

Aspergillus

spp

PSC18 1

2

3

4

5

45,000

38,700

36,800

37,600

33,400

55,100

49,100

47,600

45,400

43,100

5,000

2,600

2,500

2,320

2,800

14,480

13,512

13,311

12,611

12,302

Fusarium spp

PUE6 1

2

3

4

5

45,000

36,200

32,300

28,400

25,600

55,100

44,200

43,000

41,600

38,400

5,000

4,000

2,200

1980

1,800

14,480

12,781

11,821

11,412

11,121

Mucor spp

Keys; BOD= Biological oxygen demand, COD= Chemical oxygen demand, TSS= Total

suspended solids.

Fig 1: BOD reduction by isolates from samples

Days



59

Key: PCE1: Bacillus spp PSC1: Micrococcus spp PSC3: Pseudomonas spp

PSC6: Aeromonas spp PSU18: Geotricum spp PCE5: Aspergillus spp

PSC18: Fusarium spp PUE6: Mucor spp

Fig 2: COD reduction by isolates from samples




Fig 3: Oil and grease reduction by isolates from samples



60




Fig 4: TSS reduction by isolates from samples




Palm oil mill effluent percentage reduction potential of selected isolates from samples

Table 2 presents the result of palm oil mill effluent percentage reduction potentials of

selected isolates from samples. It showed that Pseudomonas spp (PSC3) had the highest BOP

percentage reduction (53.33%) (fig 5), and COD percentage reduction (47.37%) (fig 6).

Mucor spp (PUE6) and Fusarium spp (PSC18) had the highest oil and grease percentage

reduction (64%) (fig 7) while Mucor spp (PUE6) had the highest TSS percentage reduction

(23.20%) compared to other isolates (fig 8)



61

Table 2: Palm oil mil effluent percentage reduction potential of selected isolates from

samples

Isolate code BOD (%) COD (%) Oil and Grease (%) TSS (%) Probable organism

PCE1 16.87 26.49 24 10.91 Bacillus spp

PSC1 19.78 16.29 34 17.12 Micrococcus spp

PSC3*** 53.33 47.37 62 5.66 Pseudomonas spp

PSC6*** 34 27.22 57.24 8.15 Aeromonas spp

PSU18 22.35 17.82 36 15.67 Geotricum spp

PCE5 22.30 19.78 44 15.04 Aspergillus spp

PSC18*** 25.78 21.78 64 15.04 Fusarium spp

PUE6*** 43.11 30.31 64 23.20 Mucor sspp

Keys: ***= Potential microorganisms for biodegradation of palm oil mill effluents, TSS=

Total suspended solid, BOD= Biological oxygen demand.

Fig 5: BOD percentage reduction potential of selected isolates from samples

Fig 6: COD percentage reduction potential of selected isolates from samples



62

Fig 7: Oil and grease percentage reduction potential of selected isolates from

samples

Fig 8: TSS percentage reduction potential of selected isolates from samples

DISCUSSION

Palm mill effluent microorganisms have been known to survive in only wastewater or soils

by producing the enzyme lipase and or spores (as the case may be with fungi) which enables

them in surviving the anaerobic nature of POME (Ohimain et al., 2012). Orji et al., (2006)

stated that oily substrate provides a good environment for lipolytic microorganisms to

flourish. The biodegradation of oil in the environment is a complex process, whose

Oil

an

d g

rea

se (

%)



63

quantitative and qualitative aspects depend on nature and amount of the oil present, the

ambient and the seasonal environmental condition, and the constitution of the indigenous

microbial community (leachy and Colwell, 1990; Hinchee and Olfenbuttel, et al., 1991). This

is in compliance with this study, whereby constitution of in POME (Izah and Ohimain. 2013).

(Orji et al., 2006) stated that oily substrate provides a good environment for lipolytic

microorganisms to flourish. This is in compliance with this study, whereby constitution of

indigenous bacteria and fungi isolated from POME and its polluted soil samples were used

for degradation of palm oil mill effluent samples. The ability of the bacterial and fungi

isolates to biodegrade palm oil mill effluent was demonstrated in terms of reduction in BOD

(mg/L), COD (mg/L), TSS (mg/L), Oil and Grease(mg/L) and percentage degradation

determined from the equation earlier mentioned.

Amongst, the bacteria isolates screened for palm oil mill effluent biodegradability for 5 days,

Pseudomonas spp (PSC3) showed the highest BOD (53.33%) and COD (47.37%) while

Fusarium spp (PSC18) and Mucor spp (PUE6) showed the highest Oil and grease (64% each)

and Mucor spp (PUE6) had the highest TSS reduction potential (23.20%). In compliance with

the observation from this study, other researchers have reported biodegradation of oily

wastewaters with significant reduction in TSS, Oil and Grease and similar parameters (El-

masery et al., 2005; Raj and Murthy, 1999).

Generally, the BOD, COD, TSS and Oil and grease reduction observed is as a result of

microbial oil degradation which is considered to occur as a result of hydrolysis of oil by

secretion of lipase (oil degradation enzyme), which degrades the oil to organic acids and

Volatile Fatty Acids (VFA) or reduces it to a low molecule via beta oxidation (Fatty acid

degradation pathway) and finally the oil is decomposed to CO2 and H20 (Koshimizu et al.,

2007). Since Palm oil mill effluent contains a very high organic matter (Organic carbon) (as

evident in the result of Physicochemical analysis obtained in this study) which is generally

biodegradable, this facilitates the application of biological treatment based on aerobic process

(Chin and Wong et al., 2003). This biological treatment depends greatly on active

microorganisms, which utilizes the organic substances present in the POME as nutrients and

eventually degrades these organic matters into simple by-products such as methane,

carbondioxide, hydrogen sulphide and water (Jameel and Olanrewaju et al., 2011).



64

Thus, the exploitation of these microorganisms isolated from palm oil mill effluent for

biodegradation and bioremediation purposes will offer a very efficient tool for purifying

contaminated effluents water.

CONCLUSION

From this study, it is evident that POME degrading microorganisms can be isolated from

POME polluted area and the degrading ability of these microorganisms; Pseudomonas spp

(PSC3), Aeromonas spp (PSC6), Fusarium spp (PSC18), Mucor spp (PUE6) is a clear indicator

that they can be applied in bioremediation techniques for biodegradation of POME to

enhance treatment.

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Palm Oil Mill Effluent Biodegradation Potentials among ...ijsrm.humanjournals.com/wp-content/uploads/2017/12/5.E.O.-Eno-S.P... · 05/12/2017 · Palm Oil Mill Effluent Biodegradation